Pivotal Role of Iron in the Regulation of Cyanobacterial Electron Transport.

Iron-containing metalloproteins are the main cornerstones for efficient electron transport in biological systems. The abundance and diversity of iron-dependent proteins in cyanobacteria makes those organisms highly dependent of this micronutrient. To cope with iron imbalance, cyanobacteria have developed a survey of adaptation strategies that are strongly related to the regulation of photosynthesis, nitrogen metabolism and other central electron transfer pathways. Furthermore, either in its ferrous form or as a component of the haem group, iron plays a crucial role as regulatory signalling molecule that directly or indirectly modulates the composition and efficiency of cyanobacterial redox reactions. We present here the major mechanism used by cyanobacteria to couple iron homeostasis to the regulation of electron transport, making special emphasis in processes specific in those organisms.

Exploring the interaction of microcystin-LR with proteins and DNA.

The physiological role of microcystin-LR is still under discussion, and since binding of microcystin-LR to proteins different from their main cellular targets was described, we have performed experiments in order to explore this interaction. A non-specific interaction of microcystin-LR with a variety of soluble proteins in vitro is disrupted when using organic solvents such as methanol. The isoelectric point of proteins is not affected by their interaction with microcystin-LR, even though the presence of microcystin-LR alters the pool of peptides obtained by tryptic digestions. Under the conditions tested, microcystin-LR does not exhibit affinity for DNA. Although it is unlikely that the non-specific binding of microcystin-LR to proteins has a physiological meaning, one must be aware of the fact that determinations of the toxin extracted from any biological sample may be affected by the presence of proteins in the extracts. Consequently, we strongly recommend use organic solvents and to lyophilise the tissue samples to guarantee the accessibility of these organic solvents to microcystin-LR when performing experiments with tissue or cell extracts.

Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn(2+) are positive effectors in the binding to isiB and furA promoters.

The Fur (ferric uptake regulator) protein is a global regulator in most prokaryotes that controls a large number of genes. Fur is a classical repressor that uses ferrous iron as co-repressor and binds to specific DNA sequences (iron boxes) as a dimer. Three different genes coding for Fur homologues have been identified in Anabaena sp. PCC 7120. FurA controls the transcription of flavodoxin, the product of the isiB gene, and is moderately autoregulated. In this work, the promoter of the furA gene was defined and the FurA protected regions in the furA and isiB promoters were identified, showing that the binding sites for Anabaena FurA contain A/T-rich sequences with a variable arrangement compared to the conventional 19-base pair Fur consensus. The influence of different factors on the interaction between FurA and the promoters was evaluated in vitro. The affinity of FurA for the DNA targets was significantly affected by the redox status of this regulator and the presence of Mn(2+). The optimal binding conditions were observed in the presence of both Mn(2+) and DTT. Those results suggest that, in addition to iron availability, FurA-DNA interaction is modulated by redox conditions.

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter.

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.

Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca.

BACKGROUND: [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.

The synthesis of (R)-(+)-lipoic acid using a monooxygenase-catalysed biotransformation as the key step.

2-(2-Acetoxyethyl)cyclohexanone (4) was converted into the lactone (-)-(5) regio- and enantioselectively using 2-oxo-delta 3-4,5,5-trimethylcyclopentenyl acetyl-CoA monooxygenase, an NADPH-dependent Baeyer-Villiger monooxygenase from camphor grown Pseudomonas putida NCIMB 10007. The lactone (-)-(5) was converted into (R)-(+)-lipoic acid in six steps. In contrast cyclopentanone monooxygenase, an NADPH-dependent Baeyer-Villiger monooxygenase from cyclopentanol-grown Pseudomonas sp. NCIMB 9872 selectively oxidized the (S)-enantiomer of the ketone (4) giving better access to optically enriched, naturally occurring lipoic acid.

X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain.

BACKGROUND: Immunoglobulin domains owe a crucial fraction of their conformational stability to an invariant central disulfide bridge, the closure of which requires oxidation. Under the reducing conditions prevailing in cell cytoplasm, accumulation of soluble immunoglobulin is prohibited by its inability to acquire and maintain the native conformation. Previously, we have shown that disulfide-free immunoglobulins can be produced in Escherichia coli and purified from cytoplasmic extracts. RESULTS: Immunoglobulin REIv is the variable domain of a human kappa light chain. The disulfide-free variant REIv-C23V/Y32H was crystallized and its structure analyzed by X-ray crystallography (2.8 A resolution). The conformation of the variant is nearly identical to that of the wild-type protein and the conformationally stabilized variant REIv-T39K. This constitutes the first crystal structure of an immunoglobulin fragment without a disulfide bridge. The lack of the disulfide bridge produces no obvious local change in structure (compared with the wild type), whereas the Y32H mutation allows the formation of an additional hydrogen bond. There is a further change in the structure that is seen in the dimer in which Tyr49 has flipped out of the dimer interface in the mutant. CONCLUSIONS: Immunoglobulin derivatives without a central disulfide bridge but with stringently conserved wild-type conformation can be constructed in a practical two-step approach. First, the protein is endowed with additional folding stability by the introduction of one or more stabilizing amino acid exchanges; second, the disulfide bridge is destroyed by substitution of one of the two invariant cysteines. Such derivatives can be accumulated in soluble form in the cytoplasmic compartment of the E. coli cell. Higher protein yields and evolutionary refinement of catalytic antibodies by genetic complementation are among the possible advantages.

The covalent linkage of a viologen to a flavoprotein reductase transforms it into an oxidase.

Chemical cross-linkage of the positively charged viologen N-methyl-N'-(aminopropyl)-4-4'-bipyridinium dibromide (APMV) to the enzyme ferredoxin-NADP+ reductase from the cyanobacterium Anabaena PCC 7119 has been performed using the carbodiimide 1-ethyl[3-(3-dimethylaminopropyl)]carbodiimide. 0.5-1 mol, depending on the preparation, is introduced for each mol enzyme. The residue involved in the covalent linkage with the viologen, Glu139, has been identified using HPLC separation of the modified proteolytic peptides and subsequent sequencing. Modification of the enzyme changes its catalytic specificity since it is able to react directly with oxygen; this is observed by a high NADPH oxidase activity, which is completely absent in the native enzyme. More important, this new enzymic activity is indicative of the intramolecular electron transfer between the natural redox cofactor FAD and the artificially introduced viologen. Electrons can also flow in the reverse direction, from the viologen to the FAD group, then to NADP+, when the reaction is performed using glassy-carbon electrodes to reduce the viologen. Cyclic voltammetry experiments have shown that there is a small catalytic current between the electrode and the enzyme which is not observed in the native enzyme.

Interference of nucleases in cyanobacterium ferredoxin purification.

Isolation of cyanobacterial ferredoxin is normally carried out using nucleases in order to degrade the nucleic acids that accompany this protein during the purification procedure. However, this practice presents the inconvenience that these proteins remain in trace amounts in the purified ferredoxin preparations, although they are not visible by electrophoretical techniques. Evidence of that fact is shown in this report and an alternative procedure is described for the rapid preparation of ferredoxin from crude extracts of Anabaena PCC 7119. The method involves a treatment of the crude extract with streptomycin sulphate, a high molecular weight polication that precipitates the nucleic acids in the beginning of the purification.

Transient kinetics of intracomplex electron transfer in the human cytochrome b5 reductase-cytochrome b5 system: NAD+ modulates protein-protein binding and electron transfer.

Transient kinetics of reduction and interprotein electron transfer in the human cytochrome b5 reductase-cytochrome b5 (b5R-b5) system was studied by laser flash photolysis in the presence of 5-deazariboflavin and EDTA at pH 7.0. Flash-induced reduction of the FAD cofactor of b5R by deazariboflavin semiquinone (in the absence of b5) occurred in a rapid second-order reaction (k2 = 3.1 x 10(8) M-1 s-1) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome b5 (in the absence of b5R) was also rapidly reduced in this system with k2 = 3.1 x 10(8) M-1 s-1. When the two proteins were mixed at low ionic strength, a strong complex was formed. Although the heme of complexed b5 could be directly reduced by deazariboflavin semiquinone, the second-order rate constant was nearly an order of magnitude smaller than that of free b5 (k2 = 3.4 x 10(7) M-1 s-1). In contrast, access to the FAD of b5R by the external reductant was decreased by considerably more than an order of magnitude (k2 < 1 x 10(7) M-1 s-1). When an excess of b5R was titrated with small increments of b5 and then subjected to laser flash photolysis in the presence of deazariboflavin/EDTA, interprotein electron transfer from the b5R FAD semiquinone to the heme of b5 could be observed. At low ionic strength (I = 16 mM), the reaction showed saturation behavior with respect to the b5 concentration, with a limiting first-order rate constant for interprotein electron transfer k1 = 375 s-1, and a dissociation constant for protein-protein transient complex formation of approximately 1 microM. The observed rate constants for interprotein electron transfer decreased 23-fold when the ionic strength was increased to 1 M, indicating a plus-minus electrostatic interaction between the two proteins. Saturation kinetics were also observed at I = 56, 96, and 120 mM, with limiting first-order rate constants of 195, 155, and 63 s-1, respectively. In the presence of NAD+, the transient protein-protein complex was stabilized by approximately a factor of two, and limiting first-order rate constants of 360 s-1 were obtained at both I = 56 mM and I = 96 mM and 235 s-1 at I = 120 mM. Thus, NAD+ appears to stabilize as well as to optimize the protein-protein complex with respect to electron transfer. Another effect of NAD+ is to appreciably slow autoxidation and disproportionation of the FAD semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)